Officers in Fife Constabulary are probing the historic claim, which was made by a woman more than 20 years ago.
Walker was expelled from the SNP earlier this month after this newspaper revealed that his three former wives had accused him of attacking them.
One of the wives said he beat her with a metal coat-hanger in a hotel room in San Diego, while another spouse alleged various assaults during their time together.
His former step-daughter alleged Walker, who is over 6ft tall, hit her on the head with a saucepan when she was a teenager. She also said Walker smacked her on the "bare bottom" when she was 15.
It was then revealed that Fife Constabulary's Public Protection unit was "making enquiries" into the wife-beating claims, which span four decades.
The force has contacted all three ex-wives, one of whom is expected to be interviewed in the next few days. Another former wife has already spoken to detectives.
As part of the probe, detectives are examining an allegation that the Dunfermline MSP raped a female decades ago.
Despite being expelled by the SNP, a defiant Walker is refusing to quit the Scottish Parliament. In an interview with the Dunfermline Press, he said: "I've had a huge amount of support. I'm considering appealing [the SNP decision] but the short answer is I don't know yet.
"People have been saying they are disgusted by the way I've been treated by the party. These are councillors, members of the party and ordinary people in and around Dunfermline and Fife.
He also complained about "stuff that's been dredged up, allegations from 20, 30, 40 years ago".
Walker said of his decision not to stand down and trigger a by-election: "There have been calls for me to resign but I've never known an MSP or MP to ever do that and I'm certainly not going to set a precedent."
Labour and the Tories have called for Walker to stand down, while Scottish Liberal Democrat leader Willie Rennie said the MSP should "think about whether he should continue".
The SNP is expected to make a statement after a potential appeal by Walker against expulsion has been heard.
A spokesman for Fife Constabulary declined to comment about the rape allegation.
Labour MP for Dunfermline Thomas Docherty said: "These are extremely distressing reports. Rape is one of the most horrific crimes imaginable, and the list and severity of allegations against Mr Walker grows by the day. He is completely unfit to be an MSP and must resign his seat today."
An SNP Spokesman said: "Bill Walker was expelled from the party last Saturday, subject to an appeal against the decision. As the disciplinary process is ongoing, it would be inappropriate to comment further."
Walker could not be contacted.
BY PAUL HUTCHEON